Cellular reorganization in auxin-dependent pattern formation during early embryogenesis in Arabidopsis thaliana

A fundamental question in developmental biology is how the complex cellular pattern in multicellular organisms arises from a single cell. In land plants, the biosynthesis, transport, and signaling of phytohormone auxin is essential for pattern formation in embryogenesis. In Chapter 1, a brief introduction on plant embryogenesis, the roles of auxin signaling in pattern formation in early embryo, cellular basis on oriented cell division, and auxin-regulated oriented cell division during early embryogenesis are described as the foundation of this thesis aimed to answer the domain and cellular structures regulated by auxin that lead precise pattern formation during early embryo development of Arabidopsis thaliana. In Chapter 2, two novel fluorescent protein-based reporters for auxin perception and response, respectively, were developed to overcome technical bottlenecks for dissecting auxin signaling in embryos. The novel reporters offer higher sensitivity and responsiveness compared to existing tools. Our reporters revealed the gradients and maxima of auxin perception and response that had been hypothesized, but not yet detected. In addition, these new tools now offer a wider scope of application beyond the embryo and are generic tools for the auxin biology research community. In Chapter 3, the auxin reporters described in the previous chapter were improved to overcome their limitations, and the first comprehensive auxin reporter that was able to simultaneously visualize both auxin perception and response was characterized. With this new auxin reporter, the differential auxin signaling capacity between different cell types and differentiation states was demonstrated. In addition, the reporter for auxin response described in the previous chapter was applied in mutant embryos with a local auxin response defect, revealing its broad impact on auxin output. In Chapter 4, a toolkit of fluorescent protein-based markers labeling specific cellular structures was established. The structures included the plasma membrane, cytoskeletons, organelles, and the nucleus, structures excepted to participate in the oriented cell divisions that shape the early embryo. Expression of the protein markers was optimized for early Arabidopsis embryos, and topologies of subcellular structures were mapped during cellular reorganization in early embryogenesis. In addition, a specialized imaging technique was developed to allow high-resolution 3-dimensional imaging within the special embryo geometry. Combining the embryo-specific cellular structure marker set and the optimized imaging approach, 3-dimentional imaging of cellular structures in early embryos was achieved, and the dynamic organizations of organelles and cytoskeletons along with the unexpected discovery of early establishment of central/peripheral polarity in early embryos are described in this chapter. In Chapter 5, part of the toolkit established in the previous chapter was applied to embryos with inducible suppression on auxin response. Previously, it was shown that suppression of auxin response leads to divisions that follow only the cellular geometry, while auxin response allows cells to divide asymmetrically by deviating from this mode. It was unclear if and how the cytoskeleton mediates this auxin output, which was tested by visualizing the effect of auxin response on cytoskeleton organization. Distinct effects on both actin and microtubule properties were identified, and this provides an indication for further investigation into the biochemical and biomechanical mechanisms of pattern formation. In Chapter 6, the discoveries described in this thesis are placed in a broader context and discussed along with the latest technological and scientific advances related to the topic to offer future perspective in understanding the mechanisms underlying pattern formation.